Biochemical testing for research and diagnostic applications can require simultaneous assays including a large number of analytes in conjunction with one or a few samples and can include extended sample manipulation, multiple test substrates, multiple analytical instruments, and other steps. It is desirable to provide a method for analyzing one or a few biological samples using a single test device with a large number of analytes while requiring a small amount of sample. It is desirable to provide a device that is small in size while providing high-sensitivity detection for the analytes of interest with minimal sample manipulation. It is desirable to provide a method of loading the sample(s) into chambers on the substrate and individually sealing each chamber. It is further desirable to provide a mechanism for venting of the substrate during filling, while also avoiding and/or minimizing leakage of fluid (e.g., biological sample and/or reagents) from the test device.